108
J . Org. Chem., Vol. 36, No. 1, 1971
WHISTLER,DONER, AND NAYAK
scale as above) were: ethyl acetate, 0.9; carbon tetrachloride, 1.6. Determination of +,(aens).-To determine the sensitized quantum yield of IIb formation (+r(sensi), the Bausch and Lomb monochrometer apparatus was used. The grating was set at 366 mp and the exit slit was opened to 3.4 mm. A 70-min irradiation of 60 ml of standard actinometry solution (done before and after the reaction) gave about 5% decomposition of oxalic acid. The initial intensity was 4.03 X 1016 hvlsec and the final intensity was 4.15 X 10l6hvlsec for an average of 4.09 AZ 0.06 X 10l6hvlsec. For the reaction, 2.656 g of coumarin (0.303 M ) , 1.110 g of benzophenone (0.101 M ) , and 60 ml of carbon tetrachloride were degassed for 1hr with nitrogen and then irradiated (with stirring) for 8 hr. Benzophenone absorbs all of the incident energy at this wavelength and concentration (at 366 mp qaoo 50; 0.11). The IIb isolated was 173 mg (0.593 mmol), thus giving +r(eens) = 0.30 Determination of Relative Values.-This determination was made using the 450-W mercury arc lamp-turntable system. A corex filter was used and soft glass tubes were irradiated for 3 hr. Table X shows the initial contents of the tubes and the amounts of IIb isolated after the reaction.
RESULTS OF
TABLE X (RELATIVE) EXPERIMENT Coumarin, Benzophenone,
Solvent
g
g
IIb, g
Toluene 2.215 (0.012)a Toluene 2.215 0.0496 0.842 Ethyl acetate 2,250 (0.004) Ethyl acetate 2.225 0.0489 0.272 Acetonitrile (0.004) 2.225 0.086 2.237 0.0491 Acetonitrile 2.219 Carbon tetrachloride 0.028 1,090 2.192 0.0497 Carbon tetrachloride a Parenthesized values are estimates based on the carbon tetrachloride value of 28. From the amounts of IIb formed and the +r and +r(sens) values for carbon tetrachloride, it was calculated that benzophenone absorbed 0.012 einsteins and coumarin absorbed 0.27 einsteins of light during the irradiation.
Determination of +lr(1rb) .-The ap aratus for this experiment was the Rayonet reactor with 3500 {lamps and the quartz well with NiSOl and Pyrex filters. Coumarin absorbs >99.88% of the incident light under the conditions of the reaction which were 13.16 g of coumarin (0.30 M ) , 0.337 g of benzophenone (0.006 M ) , and 300 ml of ethyl acetate. The irradiation time was 62 hr, 25 min. The initial intensity was 2.31 X 10'7 hv/sec and the final intensity was 2.49 X 1017 hvlsec, for an average of 2.40 f 0.9 X 1017 hvlsec. The IIb isolated was 34 mg (1.16 x 10-4 mol), $'r(IIb) = 1.3 X Comparable experiments were run with carbon tetrachloride and toluene as solvents. The data here were 9 mg of IIb (+'r(IIb) = 3.1 X and 13 & 3 mg of IIb (+'r(IIb) = 2 =k 1 X 10-9, respectively. Determination of + i o for Coumarin, Trace of Benzophenone Present.-The apparatus for this experiment was the Rayonet reactor (with 3500 lamps) and the quartz well. The reaction mixtue contained 13.184 g of coumarin (0.30 M ) , 2.049 g of cis-piperylene (0.10 M ) , 1.3 ml of n-hexane, 0.349 g of benzophenone (0.006 M ) , and 300 ml of ethyl acetate. Since the intensity of the lamps in this system was 2.4 X 10l7 hvlsec in every previous determination, this value was assumed. The irradiation was carried out for 95 hr giving a total energy absorbed by coumarin of 0.136 einsteins. Initially the ratio of standard to piperylene was 1.00:2.44. After irradiation, the ratio in two trials was 1.00:2.42, showing no loss of piperylene. The per cent of trans isomer found in two trials was 3.5 and 3.7% for an average of 3.6 f 1% trans (1.08 X mol) which implies quenching of 1.96 X mol of coumarin triplets. Thus +io was found to be 1.4 X lo-+.
Registry No.-I, 91-64-5; 5248-12-4.
IIa, 5248-11-3;
IIb,
Acknowledgments. -We are grateful to the Public Health Service (Grant Yo. CA-10733, National Cancer Institute) and the Purdue Research Foundation (David Ross Fellowship to R. H., 1967-1969) for generous support of this research. We also wish to thank Dr. Angelo Lamola for kindly making his emission data available to us.
4-Thio-~-arabinofuranosylpyrimidine Nucleosides1 ROYL. WHISTLER,"LANDISW. DONER, AND U. G. NAYAK Department of Biochemistry, Purdue University, Lafayette, Indiana 47907 Received February 13, 1970 Reaction of 2,3,5-tri-O-benzoyl-4-thio-p-~-arabinofuranosyl bromide (11) with the appropriate trimethylsilylated pyrimidine has led to the preparation and isolation of the O!-D and p-D foims of 1-(4-thio-~-arabinofuranosy1)uracils (Va and Vb) and the p-D forms of thymine VIIb and cytosine IXb. The synthesis of IXb has also been accomplished by ammonolysis of l-(2,3,5-tri-0-benzoyl-4-thio-p-n-arabinofuranosyl)-4-th~ourac~l
W). 1-(p-D-Arabinofuranosyl)cytosine has been shown to possess significant carcinostatic as well as a broad spectrum antiviral act,ivity4-$ in vitro against
* T o whom correspondence should be addressed. (1) This work was supported by t h e Iiational Institutes of Health, Education and Welfare Grant KO,TROI AM 11463, Journal Paper S o . 3979, of t h e I'urdue Agricultural Experiment Station, Lafayette, I n d . 47907. (2) H. E. Skipper, F. XI. Schahel, Jr., and IT-, S.Wilcox, Cancer Chemother. Rep., 51, 125 (1907). ( 3 ) R . J. Payac, J . .Vat. CancerInst., 40, 997 (1968), and footnotes therein. (4) H . E . Renis, C. A . Holloi~ell,and G . E. Underwood, J . Med. Chem., 10, 777 (1967), and footnotes therein. ( 5 ) L).A . Buthala, Proc. Soc. E r p . B i d . Med., 115, 60 (1961). (6) J . Levitt and Y . Becker, Virology, 3 1 ( 1 ) , 129 (1967). (7) I f . A . Chirigos, M e t h o d s Drug Eual., Proc. Int. Szjmp., Milan, 3S2 (1965).
DNA viruses, Here we describe t,he preparation of the nucleoside analog in which the ring oxygen of the sugar moiet'y is replaced by a sulfur atom. The syntheses of 1-(4-thio-p-~-a~rabinofuranosyl)thymine (VIIb) and bot'h anomers of 1-(4-t~hio-~-arabinofuranosyl)uracil (Va and Vb) are also described. The synthesis of met~hyl4-thio-/3-~-arabinofuranoside from D-glUCOse has been reported.10 This compound is ( 8 ) R. A . Hyndiuk, H. E. Kaufman, E . Ellison, and Y . Centifanto, Chemotherapy, 13, 139 (1968). (9) E . C. IIerrmann J r . , A p p l . Microbial., 16(S), 1151 (1908). (10) (a) R . L . Vhistler, U. G. Kayak, and A . K.Perkins, Jr., Chem. Gommun., 21, 1339 ( 1 9 6 8 ) ; (b) U. G. Nayak and R . 1,. V'histler, J . OW. Chem., 85, 519 (1970).
4-THIO-D-ARABINOFURANOSYLPYRIMIDINE NUCLEOSIDES
J . Org. Chew., Vol. 36, No. 1 , 1971 109
converted to 2,3,5-tri-O-benzoy1-4-thio-,8-~-arabinofu-instrument. Evaporations were done under diminished pressure with a bath temperature below 40". Absorption chromatography ranosyl bromide (11) according to a procedure given by was made on silica gel.'* Optical rotations were measured on a Ness and Fletcher.ll Assignment of the P-D configuraPerkin-Elmer Model 141 polarimeter. tion to the major product formed is based on its being Methyl 2,3,5-Tri-0-benzoyl-4-thio-p-~-arabinofuranoside (I).more levorotatory than the minor product (Y-D-111. To a stirred, ice-cooled solution of methyl 4-thio-p-D-arabinofuranoside (9 g, 0.05 mol) in 100 ml of dry pyridine was added Also, the greaker coupling constant J for the P-D anomer benzoyl chloride (23.26 g, 0.165 mol) dropwise over a period of is consistent with the cis relationship of the hydrogen 30 min. Benzoylation was complete in 2 hr as monitored by tlc atoms on C-1 and C-2. in solvent A. The reaction mixture was poured into 600 ml of Displacement of bromide ion from I1 by trimethylice and water under stirring and stirring continued for 2 hr at which time the mixture was extracted with 500 ml of chloroform. silyl derivatives12 of uracil and thymine leads to the forThe chloroform extract was washed sequentially with water, 2 mation of anameric mixtures of the benzoylated nucleoN hydrochloric acid until slightly acidic, 1 S sodium hydroxide sides, which are separated chromatographically. Nishsolution until slightly alkaline, and water until neutral. The imura and Shimizu13 have used similar procedures in chloroform solution so washed was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under ditheir synthesis of the anomeric pyrimidine nucleosides minished pressure to obtain 25 g of pure syrup methyl 2,3,5-triof D-arabinofuranose. Debenzoylation is achieved by O-benzoyl-4-thio-p-~-arabinofuranoside (I): [CY]25D - 174.3" (c treatment of the blocked nucleosides with sodium in 1.05, CHC13). Compound I was used directly for the preparation methanol. of the bromo sugar. 1-(4-Thio-,8-~-arabinofuranosyl)cytosine (IXb) is preAnomeric 2,3,5-Tri-0-benzoyl-4-thio-n-arabinofuranosy1 Bromides (I1 and III).-To a stirred solution of compound I (25 g) pared by two methods. Reaction of I1 with bis(triin 125 ml of glacial acetic acid was added 125 ml of 3270 (,w/w) methylsily1)-N-acetylcytosine yields a mixture of the hydrogen bromide in glacial acetic acid. The reaction mixture anomeric forms of the blocked nucleosides, from which was stirred for 20 min at 25' during which time the entire mass the 6-Danomer (VIIIb) crystallizes. The other method solidified. This was diluted with 750 ml of dry methylene chloride. The solution was poured into 2.5 1. of ice and water and of producing this nucleoside involves thiationI4 of 1organic layer was quickly washed sequentially with water, a (2,3,5-tri-O-benzoy1-4-thio-p - D - arabinofuranosyl) uracil the 107' aqueous sodium bicarbonate solution, and water. The (IVb) to its $-thiouridine analog X. This derivative, washed methylene chloride solution was dried over anhydrous upon treatment with methanolic ammonia, gives 1-(4sodium sulfate and filtered. The filtrate was concentrated under thio-P-D-arabinofuranosy1)cytosine(IXb). diminished pressure at a bath temperature of 30". The residue solidified when concentrated and was recrystallized from methIt has been observed,l5 previously, that the optical ylene chloride-hexane (75 ml: 300 ml). Needle-shaped crystals of rotatory relationship of the anomeric pyrimidine nucleo2,3,5-tri-O-benzoy1-4-thio-p-n-arabinofuranosy1 bromide (11)sepsides do not obey Hudson's isorotation rules. These arated: yield 22 g; mp 126'; [ m ] " D -171" (c 1, CHzClz). exceptions to ]Hudson's rules have been found to hold for Anal. Calcd for Cz6H21Br0&3: C, 57.67; H, 3.91; Br, 14.56; and is further the anomeric ~-arabinofuranosyluracils'~ S, 6.05. Found: C, 57.80; H, 4.03; Br, 14.58; S, 6.05. Further concentration of the mother liquors produced 5 g more supported by the optical rotatory dispersion (ORD) of I1 to give a total yield of 27 g. Additional concentration promeasurements on a large number of pyrimidine nucleoduces a small amount of the m - anomer ~ 111: Ji.2 = 4.5 cps for sides. The anomeric configuration of the nucleosides I1 and 0 cps for 111. described here cannot be assigned from ORD measureAnomeric 1-(2,3,5-Tri-0-benzoyl-4-thio-~-arab~nofuranosyl)uracils (IVa and IVb).-A stirred mixture of 5.41 g (0.01 mol) ments since both anomers show positive Cotton effects of compound I1 and 3.84 g (0.015 mol of bis(trimethylsily1)uracil in blocked and unblocked forms. Tentative anomeric was heated on an oil bath at 130-135" for 3 hr in a current of dry configurations are assigned on the basis that the 0-D nitrogen. During this time the bromo sugar I1 disappeared as anomer is expected in greatest yield. Far less signifiindicated by tlc in solvent B. The yellowish gummy product was cant is the observation that benzoylated P-D anomers refluxed for 20 min with 50 ml of 95% ethanol. After evaporation of the solvent under diminished pressure, the residue was taken have had, in our hands, the slowest chromatographic into benzene and refluxed for 5 min and filtered. The filtrate was mobilities on silica gel G in solvent C.
Experimental Section Analytical Methods.-Purity of products was determined by thin layer chromatography (tlc) on silica gel GI7 coated glass plates (5 x 13 cm) irrigated with (a) benzene-ethyl acetate (6: l ) , (b) hexane-ethyl acetate (4: l ) , (c) benzene-ethyl acetate (10: l ) , (d) chloroform-acetone (15: I ) , (e) chloroform-acetone (30: l ) , and ( f ) chloroform-acetone (9:l). Solvent ratios are based on volumes. Components were located by spraying with 570 sulfuric acid in ethanol and heating until permanent char spots were visible. Melting points were determined on a FisherJohns apparatus and are corrected. Nuclear magnetic resonance (nmr) spectra were obtained with a S'arian Associates A-60 (11) IL. K. Ness and H. G. Fletcher, Jr., J . Amer. Chem. Soc., 80, 2007 (1958). ( 1 2 ) T. Hishimura a n d I. Iwai, Chem. Pharm. Bull., 12, 352 11964). (13) T. Nishimurrt a n d B . Shimizu, ibid., 18, 803 (1965). (14) J. J. Fos, D. Van Praag, I. Wempen, I. L. Doerr, L. C'heong, J. E.
Knoll, M . L. Kidinoff, A. Bendich, and G. B. Brown, J. Amer. Chem. Soc., 81, 178 (1959). (15) J. FarkaB, L. Kaplan, a n d J. J. Fox, J . Org. Chem., a9, 1469 (1964). (16) T. R. Emerson, R. J. Swan, and T. L. V. Ulbrioht, Biochemietyy, 6, 843 (1967).
(17) L. Merck Ag, Darmstadt, Germany. Instruments Ino., 11 estbury, N. Y .
Distributors:
concentrated to a brownish yellow syrup which was applied to a silica gel column (40 X 1.5 cm) prepared in benzene. The column was eluted with 1 1. of benzene, w.hich removed the olefinic contaminants. The column was then eluted with solvent C and the nucleoside fractions collected on a fraction collector. The nucleoside fractions containing both CY-D and 0-0 anomers IVa and IT'b were combined and concentrated to a foam (1.3 g) which was dissolved in 20 ml of hot ethyl acetate. On cooling, pure 1-(2,3,5-tr~-~-benzoy~-4-th~o-p-o-arab~nofuranosy~)urac~~ (IS'b) crystallized as small needles: yield 2.1 g; mp 208-209'; [LY]'~D -13.8" (C 1, CHC1,); ORD (C 0.004, CHCla) [$]z?s +4100°, [$I247 -21,800'. Anal. Calcd for C ~ O H ~ ~ N ~ C,O62.93; ~ S : H , 4.22; N, 4.89; S, 5.60. Found: C, 63.03; H, 4.25; N, 4.87; S,5.86. The filtrate after separation of the p-D anomer IVb when examined by tlc in solvent D (plate developed three times) showed the presence of more p-D anomer (minor) and the CY-D anomer (major). By repeated silica gel chromatography using solvent E as eluent the C ~ - Danomer IVa (550 mg, 8.7%) and 300 mg more of the 0-D anomer IT'b were collected. Total yield of 1-(2,3,5tri-0-benzoyl-4-thio-~-~-arabinofuranosy1)urac~l (IVb) was 2.4 g, 42Y0. The CY-D anomer I T a was recrystallized from ethanol: mp 127-128"; [ C Y ] ~ ' D +12.3' (c 1, CHC13); ORD (c 0.004, CHC13) [dlzs? +14,900", [$I262 -29,500'. Anal. Found: C , 62.92; H, 4.40; K,4.77; S, 5.87.
Brinkman ( 1 8 ) J. T. Baker Chemical Co., Phillipsburg, N. J.
110 J . Org. Chem., Vol. 36, No. 1 , 1971
WHISTLER,DONER,AND NAYAK
1-(4-Thio-a-~-arabinofuranosyl)uracil (Va).-To a solution of acetykytosine (VIIIb): mp 226-227"; [ a I a 5 +15.8" ~ (c 1, 50 mg of sodium in 10 ml of neat methanol was added a solution CHCh); ORD (C 0.004, CHC13) [$I2e7 +19,000", [$I248 -27,700'. of compound IS'a (700 mg) in 30 ml of warm neat methanol. Anal. Calcd for C32H2708N3S:C, 62.63; H , 4.44; N , 6.85; This was stirred at 25" for 18 hr. The reaction mixture was S, 5.22. Found: C, 62.41; H, 4.42; K,6.80; S,5.43. deionized using methanol-washed Amberlite I R 120 (H+) resin l-(4-Thio-p-n-arabinofuranosyl)cytosine(IXb).--A solution of and filtered. The filtrate was concentrated under diminished compound VIIIb (1.2 g ) in 50 ml of absolute methanol was pressure to a solid which was triturated with absolute ether to saturated with dry NH3 gas at 0' and kept in the refrigerator for remove methyl benzoate and filtered. The precipitate was re2 days. The mixture was then evaporated to dryness and tritucrystallized from neat methanol to yield 266 mg of 1-(4-thio-arated with dry ether three times to remove benzamide and methyl o-arabinofuranosy1)uracil (Va): mp 214-215'; [a]2 5 ~$57.7' benzoate. The residual solid was recrystallized from dry metha(c 1, HzO); uv : ' , :A mp (e, pH) 265 (10,000, 3.2), 265 (10,700, nol to give 450 mg of 1-(4-thio-~-n-arabinofuranosyl)cytosine 7.0), 265 (9600, 9.6), 267 (8600, 14.0); ORD (C 0.004, CHCla) (IXb): mp 210-211"; [ a I z 5$143" ~ (c 1, HzO). uv A",,': mp (e, pH) 280 (12,100, 3.2), 274 (9700,7.0), 274 (10,600, 9.6), [$Izss +9740', [ $ ] z 6 1 -14,430", Anal. Calcd for CsHleNzO5S:C, 41.49; H , 4.65; N, 10.77; 275 (9600, 14). S,12.33. Found: C, 41.58; H , 4.83; K, 10.86; S, 12.11. Anal. Calcd for C9H13O4r\r3S:C, 41.69; H, 5.05; N, 16.20; l-(4-Thio-p-~-arabinofuranosyl)uracil (Vb).-The p-D anomer S,12.38. Found: C, 41.45; H, 5.07; N, 16.19; S, 12.40. was debenzoylated in a manner similar to that used for the WD1-(2,s,5-Tr~-O-benzoyl-4-thio-p-~-arab~nofuranosyl)-4-thioanomer. 1-(4-Thio-p-~-arabinofuranosyl)uracil(Vb) was crysuracil (X).-A mixture containing 6.292 g (0.011 mol) of IVb, tallized from neat methanol to yield 600 mg: mp 194-195'; 10.77 g of phosphorus pentasulfide, and 150 ml of reagent grade [aIz5D +117.8" (C 1, Hz0); uv A":, mp (e, pH) 264 (10,700, 3.2), pyridine was refluxed for 5 hr. About half of the pyridine was 264 (10,400, 7.0), 264 (11,400, 9.6), 266 (9200, 14.0); ORD (c removed under diminished pressure and the dark brown colored 0.004, CHCls) [$Ins6 +4740", [$I262 -4410". solution was poured into stirred water where compound X Anal. Found: C, 41.26; H, 4.65; K, 10.71; S,12.09. started solidifying. The solid product was filtered and the preAnomeric 1-(4-Thio-~-arabinofuranosyl)thyrnines(VIIa and cipitate was taken into chloroform and filtered from insoluble VIIb).-In a procedure similar to that described above for 1material. The chloroform solution was washed twice with water (2,3,5-tr~-0-benzoyl-4-th~o-~-arabinofuranosyl)uracils, the bromo and dried over anhydrous sodium sulfate. After filtration the sugar I1 (5.41 g, 0.01 mol) was condensed with bis(trimethylsily1)chloroform solution was concentrated to a yellow solid foam thymine (4.05 g, 0.015 mol). After chromatographic purification which was crystallized from hot ethanol to give 4.96 g of comon silica gel, 3.1 g of the anomeric 1-(2,3,5-tri-O-benzoyl-4-thio- pound X as yellow needles: mp 156-157'; [ a I a 4 D +6.4" (c 1, n-arabinofuranosy1)thymines (S'Ia and T71b) was obtained. DeCHC13). benzoylation with a catalytic amount of sodium methoxide Anal. Calcd for C3oHzdO,XzSz: C, 61.21; H, 4.11; N, 4.76; followed by deionization with methanol-washed Amberlite I R S, 10.89. Found: C, 61.37; H,4.11; K,4.89; S, 10.86. 120 (H+) resin gave the anomeric mixture of l-(4-thio-n1-(4-Thio-p-~-arabinofuranosyl)cytosine (IXb ) .-Compoimd X arabinofuranosy1)thymiries (VIIa and VIIb). Crystallization (1.178 g, 0.002 mol) was treated with 160 ml of anhydrous from neat methanol gave 0.76 g (27.7y0) of pure 1-(4-thio-p-~- methanolic ammonia (previously saturated at -5 to 0") in a arabinofuranosy1)thymine (VIIb): mp 209-210'; [a]2 5 +136' ~ sealed tube at 110-115° for 18 hr. After the tube was opened, the contents were transferred to a round-bottomed flask and (c 1, HzO); uv At: mp (e, pH) 270 (10,900, 3.2), 270 (10,400, 7.0), 270 (10,200, 9.6), 271 (8500, 14.0); ORD (C 0.004, CHC13) concentrated under reduced pressure to a solid. The solid was triturated with ether to remove benzamide and the residual solid [$I290 +10,900", [ $ ] m -22,600". Anal. Calcd for CIOHUN~O~S: C. 43.78: H. 5.14: N . 10.22: dissolved in hot methanol and decolorized with charcoal. The S,11.69. Found: C, 44.26; H, 5.05; N, 10.40; S,11.93. methanolic solution was filtered and then concentrated under Anomeric 1-(2,3,5-Tr~-0-benzoyl-4-th~o-~-arabinofuranosyl)-~diminished pressure to a solid which was recrystallized from hot acetylcytosines (VIIIa and VIIIb) .-Bis(trimethylsily1)-N-acetylmethanol to give 0.37 g of compound IXb: mp 210-211'; cytosine and the bromo sugar I1 (5.41 g, 0.01 mol) were condensed [aIz5D+143" (C 1, H20). as described for 1-(2,3,5-tri-0-benzoyl-4-thio-~-arabinofuranosy1)uracils. After chromatographic purification using solvent F , the nucleoside fractions were collected to give 1.5 g of the mixture Registry No.-I, 26527-29-73 11, 26527-30-0; 111, of the CY-Dand p-D anomers S'IIIa and T?IIb. A thin layer chroIVa, 26527-32-2; IVb 26527-33-3; Va, 26527-31-1 matogram developed five times in solvent D showed the two 26527-34-4 Vb, 26527-35-5; VIIb, 26527-36-6; V IIb, distinct anomers. Recrystallization from ethyl acetate gave 1.23 g of 1-(2,3,5-tri-0-benzoyl-4-th~o-p-n-arab~nofuranosyl)-~~- 26527-37-7 IXb, 26599-17-7; X, 26527-38-8.
